PCT Application WO 95/21252 discloses and claims peptide compositions which alter the activity of a signal-generating protein with respect to its cognate binding protein wherein the cognate protein contains at least one WD-40 region which putatively interacts with the signal-generating protein. The peptide compositions mimic the WD-40 regions, thus competing with the interaction of the cognate with the signal-generating protein. This competition results either in inhibiting the signal-generation or activating it.
One specifically exemplified signal-generating protein is protein kinase C (PKC); the illustrated cognate receptor for activated kinase C (RACK), in this case specific for beta-PKC, was designated RACK1. The gene encoding RACK1 was cloned and sequenced, showing that RACK1 contains the requisite WD-40 regions.
The above PCT application and U.S. Ser. Nos. 08/473,089, 08/477,346, and 08/487,072 further describe methods to identify additional pairs of signal-generating proteins and their cognates and methods for recognizing WD-40 sequences in the cognates. These applications also note that such interactions can be used as a system to identify additional molecules that bind the signal-generating protein by measuring the effect of candidate binding molecules on the interaction between the signal-generating protein and either its cognate per se or the polypeptide compositions that mimic the WD-40 regions of the cognate.
In U.S. Pat. No. 5,783,405, several specific peptides were identified that bind either to the signal-generating protein or to the cognate protein in a signal-affecting manner. The use of the signal-generating protein/cognate system to assay for modulators of signal transduction in assays which are independent of the purity of these participants was described. The PKC enzyme system was illustrated as a specific embodiment. In addition, peptides which reside on the signal-generating protein, as well as those which reside on the cognate or mimics thereof, were described as being useful to modulate the signal-generating interactions and biological activities which are mediated by the signal-generating interactions.
In U.S. Pat. No. 5,776,716, experiments that demonstrated the identity of a cognate protein for PKC-theta as the fyn protein were described. Since it is well established that fyn is involved in mediation of T-cell responses, it is apparent that disruption of the interaction of PKC-theta with its fyn cognate mediates the immune response. It is also apparent that PKC-theta is a mediator of the immune response. Substances which can be shown to disrupt the interaction between PKC-theta and its cognate fyn, or, as described hereinbelow, to influence the interaction of PKC-theta with any cognate also have immunomodulating activity. The identification of fyn as a PKC-theta cognate, and the consequences of interfering with this association, demonstrate that PKC-theta is a significant signaling protein involved in the immune response.
PCT Application WO 97/143038 and U.S. application Ser. No. 08/665,647, filed Jun. 18, 1996, both disclose an assay system for identifying modulators of the immune system. The contents of both these documents are expressly incorporated herein by reference. The assay system measures the effect of candidate substances on the interaction between a PKC-theta, or a fragment thereof, with its cognate and then compares the result obtained to that of a standard. The cognate comprises the relevant portion of a molecule which binds to PKC-theta, e.g. fyn, the fyn fragment fyn-3, the fyn fragment fyn-2, and the protein encoded by clone 2-10 or 2-32. Because the method takes advantage of inherent biological specificity, it can be conducted on impure preparations of the participants in the signal pathway--i.e., the above-mentioned signal-generating protein PKC-theta and its cognate receptor. The assay is conducted by assessing the interaction between the signal-generating protein and its cognate either by measuring binding directly or by measuring a physiological or metabolic effect. The measurement is made in the presence and in the absence of a candidate substance. Successful candidates which agonize the signal effect an increase in a metabolic or physiological output; antagonists effect a decrease. Both antagonists and agonists compete for binding between cognate and signal-generating protein. The interactions are disclosed as being measurable in a variety of ways, e.g. measuring translocation of said PKC-theta or fragment; measuring tyrosine phosphorylation of a 21 kD protein after OKT-3 stimulation of T-cells; measuring diminution of IL4 and/or IL5 production without affecting IFN production; measuring the binding of said PKC-theta or fragment to its cognate. The binding can be determined by means of a "two-hybrid" system. The contents of each of the documents described above are expressly incorporate herein by reference.